Function and Regulation of the ETS Transcriptional Repressor Tel-1/YAN

说明(申请人提供)：本提案是对1R01GM080372-01《ETS转录抑制因子TEL1/Yen的功能和调控》的续展申请的修订。发育程序是由转录因子驱动的，转录因子作用于顺式调节增强子元件，以协调精确的基因表达模式。转录因子Ets家族中的两个成员，抑制子Yen和激活子指向，在人类中被称为TEL1和ETS1/ETS2，作为受体酪氨酸激酶/RAS/MAPK信号通路的下游效应器，在各种类型的细胞中协调增殖和分化之间的平衡。除了ETS DNA结合基序外，Yen/TEL1和Point/ETS1/2还共享第二个保守结构域，即不育α基序(SAM)，它介导同型和异型蛋白质-蛋白质相互作用。SAM介导的相互作用在正常的细胞命运转换过程中调节网络输出，并在针对Tel1基因的染色体易位所产生的功能融合中提供致癌推动力。这一建议的目的是阐明Yen/TEL1介导的抑制机制，并了解同型SAM介导的相互作用对这些机制的贡献。由于我们正在研究的信号分子和网络在哺乳动物中具有保守的功能，而且它们的失调有助于许多白血病和实体肿瘤的致癌转化，这些研究的发现将提高我们对人类发育和疾病潜在机制的理解。目的1研究抑制子Yen被招募到染色质以调节基因表达的分子机制。我们将在染色质占据和靶基因表达调控的水平上定义严点竞争是如何表现出来的。我们将测试这一挑衅性的想法，即除了作为序列特异性DNA结合抑制因子的典型作用外，yan还作为染色质相关因子发挥作用。目的2将探索一种假设，即严氏复杂的染色质占有率反映了一种缓冲基因表达的新机制，以抵御遗传和环境噪声。利用最先进的活体分子遗传学技术，我们将确定在最优条件下以及面对遗传或环境变异时，Yen结合区对调控其靶基因表达的个体贡献。利用3C和CHIP-qPCR，我们将研究三维染色质相互作用对Yen占位和基因表达调控的影响。目的3探讨主动抑制的分子机制。综合运用分子遗传学、生物信息学和生物化学的方法，我们将确定Yen转录复合体的组成，它们决定了Yen单体和聚合物的差异抑制活性，并探索聚合程度如何影响体内的抑制活性。

DESCRIPTION (provided by applicant): This proposal is a revision of renewal application for 1R01GM080372-01 "Function and regulation of the ETS transcriptional repressor TEL1/Yan". Developmental programs are driven by transcription factors that act upon cis-regulatory enhancer elements to coordinate precise patterns of gene expression. Two members of the conserved ETS family of transcription factors, a repressor Yan and an activator Pointed, known as TEL1 and ETS1/ETS2 in humans, act as downstream effectors of the receptor tyrosine kinase/Ras/MAPK signaling pathway to orchestrate the balance between proliferation and differentiation in a variety of cell types. In addition to the ETS DNA binding motif, Yan/TEL1 and Pointed/ETS1/2 share a second conserved domain, the sterile alpha motif (SAM), which mediates homo- and hetero-typic protein-protein interactions. SAM-mediated interactions modulate network output during normal cell fate transitions and provide an oncogenic driving force in the functional fusions produced by chromosomal translocations targeting the Tel1 locus. The goals of this proposal are to elucidate the mechanisms of Yan/TEL1-mediated repression and to understand the contribution of homotypic SAM- mediated interactions to these mechanisms. Because the signaling molecules and networks we are studying have conserved functions in mammals, and because their dysregulation contributes to oncogenic transformation in a number of leukemia and solid tumors, the discoveries resulting from these investigations will improve our understanding of mechanisms underlying human development and disease. Aim 1 will investigate the molecular mechanisms by which the repressor Yan is recruited to chromatin to regulate gene expression. We will define how Yan-Pointed competition manifests at the level of chromatin occupancy and regulation of target gene expression. We will test the provocative idea that in addition to its canonical role as a sequence-specific DNA binding repressor, Yan also acts as a chromatin associated factor. Aim 2 will explore the hypothesis that Yan's complex chromatin occupancy profile reflects a novel mechanism for buffering gene expression against genetic and environmental noise. Using state of the art in vivo molecular genetics technologies, we will determine the individual contribution of Yan-bound regions to regulating the expression of its target genes under both optimal conditions and in the face of genetic or environmental variation. Using 3C and ChIP-qPCR, we will investigate the influence of three-dimensional chromatin interactions on Yan occupancy and regulation of gene expression. Aim 3 will investigate the molecular mechanisms of active Yan-mediated repression. Using an integrative combination of molecular genetics, bioinformatics and biochemical approaches, we will identify the components of Yan transcriptional complexes that determine the differential repressor activity of Yan monomers versus polymers and explore how the extent of polymerization impacts repressive activity in vivo.